Abstract. The glycopeptide antibiotics are the most important class of drugs used in the treatment of resistant bacterial infections, including methicillin-resistant S. aureus (MRSA). In fact, even after 60 years of clinical use, vancomycin administration in the clinic is still steadily increasing. Consequently, the emergence of resistant Gram-positive pathogens including vancomycin-resistant Enterococci (VRE) and vancomycin-resistant S. aureus (VRSA) presents a serious public health problem at a time few new antibiotics are being developed. These two pathogens rank 4th and 5th on the WHO global priority list of antibiotic-resistant bacteria for which there is an urgent need for new treatments. The only clinically significant mechanism of vancomycin resistance is its induced late stage remodeling of the bacterial cell wall precursor termini from D-Ala-D-Ala (the target of vancomycin) to D-Ala-D-Lac. Objectives of the work have included the redesign of vancomycin for dual D-Ala-D- Ala and D-Ala-D-Lac binding capable of treating both sensitive and vancomycin-resistant bacterial infections and directly addressing the underlying molecular basis of vancomycin resistance. The exciting results with binding pocket analogs designed for dual D-Ala-D-Ala/D-Lac binding and subsequently with their peripherally modified derivatives that incorporate synergistic second and third mechanisms of action (MOAs) independent of D-Ala-D- Ala/D-Lac binding chart a compelling path forward for the development of potent and especially durable antibiotics not prone to eliciting resistance for treatment of deadly vancomycin-resistant and multidrug-resistant bacterial infections. The studies in the last grant period have produced analogs worthy of comprehensive preclinical evaluation and the challenges for the work have returned to those of their preparation. This is an area where the PI and his group are well equipped and excited to address. The proposed studies will improve access to the analogs through development of an innovative next generation total synthesis or semisynthetic approach, and lay a foundation for fermentation access to the pocket modified glycopeptide antibiotics. The immediate target of the next generation synthetic studies, which are at an advanced stage, is the synthesis of an analog that is projected to be the most active compound in the series examined to date, bearing the most effective pocket modification and two key peripheral modifications. A well-conceived stereochemical simplification in the target structures will also be examined that will substantially improve synthetic access to the aglycon core structure without compromising antimicrobial activity. Optimization of activity derived from a new, third MOA discovered in the last grant period will be conducted and such efforts have already improved on the impressive activity reported to date. In vivo assessments of key compounds will be conducted that build on the stunning results to date, structural characterization of pocket analogs bound to model ligands will be pursued to confirm the fundamental basis of the remarkable dual D-Ala-D-Ala/D-Lac binding, and a breakthrough discovery for achieving antimicrobial activity against Gram-negative as well as Gram-positive bacteria will be examined.